The pre-mRNA transcripts of most mammalian genes contain multiple introns that must be precisely removed before the mRNA can be used in protein synthesis. For many genes, this pre-mRNA splicing reaction is regulated by developmental or environmental signals. Errors in the splicing pathway can have devastating health consequences and account for approximately 15% of all known human genetic disorders. The diagnosis and treatment of splicing-related disorders will profit by a better understanding of the cellular rules governing intron removal. The pre-mRNA splicing enzyme, or spliceosome, is a large ribonucleoprotein complex composed of over 70 proteins and 5 small nuclear RNAs. A widely cited model of spliceosome assembly, activation, and disassembly (or the spliceosome cycle) has evolved from the results of in vitro experimentation conducted over the last 15 years. The modulation of splicing efficiency and alterative splice site selection often can be explained by changes in spliceosome assembly observed in the presence or absence of specific regulator proteins (or cis-acting elements). The recent isolation from yeast of a U2,U5,U6 ribonucleoprotein (RNP) complex reminiscent of a late stage or post-catalytic spliceosome supports this spliceosome cycle model. In contrast, the identification of what appears to be a fully assembled but pre-mRNA-free "penta-snRNP" spliceosome from yeast raises the question of whether spliceosome disassembly actually occurs in vivo. We will address this apparent discrepancy by tests of two hypotheses. First, that a spliceosome cycle occurs within cells and progresses through a series of splicing factor-dependent assembly and disassembly steps. Second, that the Clf1p assembly factor facilitates integration of the prespliceosome with the U4/U6.U5 tri-snRNP particle through branchpoint reorganization that includes the recruitment of RNA export factors. This work will provide new experimental tools for the analysis of RNA processing and generate conceptual frameworks from which to view spliceosome assembly and the integration of the pre-mRNA splicing and RNA export pathways in living cells.